Faculty Bibliography

In this report, the contribution of PKR to the IFN-gamma mediated inhibition of VSV replication in neurons was examined. IFN-gamma treatment of NB41A3 murine neuroblastoma cells resulted in the reduced expression of VSV protein during infection. PKR was found to be modestly upregulated in NB41A3 cells following IFN-gamma treatment. The phosphorylation state of PKR and its downstream target, eIF2alpha, were unaffected by either IFN-gamma or VSV infection. Inhibition of PKR through the use of 2-aminopurine or the expression of the Influenza A NS1 gene had no effect on the ability of IFN-gamma to inhibit the replication of VSV in vitro. These data indicate that endogenously expressed PKR is not required for the IFN-gamma mediated inhibition of VSV replication in NB41A3 neuroblastoma cells.

The innate immune system is multifaceted, comprised of preformed factors, cells, and many proteins and lipid mediators produced by those cells. In the CNS these are critical in initiation and amplification of the inflammatory response and in the subsequent elicitation of the specific T cell response to viral encephalitis. Cells that are resident in brain parenchyma and peripheral cells that are recruited both play key roles in the hosts's responses. Unlike the peripheral compartments, in the CNS, non-cytolytic means of eliminating viral infections have been critical, since, in contrast to columnar epithelial cells, neurons are non-renewing. When the innate immune responses are inefficient or absent in viral encephalitis, pathology is more likely. Much more work remains to elucidate all of the critical cells and their mediators, as well as to develop new therapies for infections of the CNS.

Interferon (IFN)-gamma, is not only a marker of T(H)1 CD4, CD8 and natural killer (NK) cells, it is also a critical antiviral mediator which is central to the elimination of viruses from the CNS. In this review, we describe IFN-gamma, its receptor, signal transduction from receptor engagement, and antiviral downstream mediators. We demonstrate that although neurons are post-mitotic and non-renewing, they respond to IFN-gamma in a fashion similar to peripheral fibroblasts or lymphocytes. We have illustrated this review with details about studies on the role(s) of IFN-gamma in the pathogenesis of measles virus (MV), herpes simplex virus (HSV) type 1, and vesicular stomatitis virus (VSV) infections of the CNS. For VSV infection, IFN-gamma signals through Jaks 1 and 2 and STAT1 to activate (interferon regulatory factor) IRF-1; although viral protein synthesis is inhibited, PKR is not a critical mediator in the antiviral response to VSV in murine neurons. In contrast, induction of nitric oxide synthase (NOS) type 1 and its production of nitric oxide is essential in the elimination of viruses from neurons.

In this report, the role of STAT4 and local production of interleukin (IL)-12 in the central nervous system (CNS) were examined during experimental vesicular stomatitis virus (VSV) encephalitis. We have previously shown that IL-12 treatment is beneficial both in vitro and in vivo during experimental VSV infection. This inhibition of VSV replication was dependent on the production of nitric oxide (NO) by the neuronal isoform of nitric oxide synthase (NOS-1). In vitro, IL-12 induces the phosphorylation and nuclear localization of STAT4 in neuroblastoma cell lines. STAT4 expression was not required for host survival or clearance of virus during experimental VSV encephalitis. Taken together, these data suggest that while neurons can respond directly to IL-12 in vitro by signaling through STAT4, STAT4 is not required for survival. It is likely that redundant innate host inflammatory cytokine responses compensate for the absence of IL-12 signaling.

In this report, the role of nitric oxide synthase (NOS) and IL-12 administration in inhibition of vesicular stomatitis virus (VSV) from infected neuroblastoma cells was examined. We previously have shown that cytokine treatment of cells results in the induction of NOS-1, and this is associated with a 2 log inhibition of VSV production. We performed these studies to examine the mechanism by which viral replication is suppressed. Neuroblastoma cells (NB41A3) were treated with either IL-12 or medium and subsequently infected with VSV. Viral protein and mRNA were isolated from these cells, and their levels were measured by Western or Northern blots, respectively. mRNA levels were decreased modestly, but viral proteins were decreased substantially in cells pretreated with IL-12, suggesting that the inhibitory effect of NO is working at the translational level. Cytokine treatment of cells was not associated with oxidative stress. The viral proteins also were nitrosylated. These data suggest that the mechanism of NO inhibition of viral replication occurs through translational interference and posttranslational modifications of viral components.